A METHOD FOR THE DETERMINATION OF 5,6-EET USING THE LACTONE AS AN INTERMEDIATE IN THE FORMATION OF THE DIOL

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biologi cal activities but the functional significance of this labile eicosano id is unknown due, in part, to difficulties of quantitation in biologi cal samples. We have developed a sensitive and specific method to meas ure 5,6-EET utilizing its selective capacity to form a lactone. The in itial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6- DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconvers ion to 5,6-DHT and quantitation by gas chromatography-mass spectrometr y (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-l actone and 5,6-DHT with a t(1/2) approximate to 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak w ith the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via c onversion to 5,6-DHT, was approx. 6-fold less than that reported for p rostaglandin E-2 (PGE(2)) and 20-HETE. The coronary perfusate concentr ation of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and c oronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.